Beijing Genotype of
            <i>Mycobacterium tuberculosis</i>
            Is Significantly Associated with High-Level Fluoroquinolone Resistance in Vietnam

China is one of the countries with the highest burdens of multidrug-resistant (MDR) and fluoroquinolone (FQ)-resistant tuberculosis (TB) globally. Nevertheless, knowledge about the prevalence and molecular characterization of FQ-resistant Mycobacterium tuberculosis isolates from this region remains scant. In this study, 138 M. tuberculosis isolates determined by the agar proportion susceptibility method to be resistant to ofloxacin (OFX) were enrolled from a national drug resistance survey of China. All these strains were tested for susceptibility to ofloxacin, levofloxacin, moxifloxacin, gatifloxacin, and sparfloxacin using liquid Middlebrook 7H9 medium. The entire gyrA and gyrB genes conferring FQ resistance were sequenced, and spoligotyping was performed to distinguish different genotypes. Overall, the prevalence of resistance in China was highest for ofloxacin (3.76%), intermediate for levofloxacin (3.18%) and moxifloxacin (3.12%), and lowest for sparfloxacin (1.91%) and gatifloxacin (1.33%). Mutations in the gyrA gene were observed in 89 (64.5%) out of the 138 OFX-resistant M. tuberculosis strains. Positions 94 and 90 were the most frequent sites of mutation conferring FQ resistance on these strains, accounting for high-level FQ resistance. Furthermore, the Beijing genotype showed no association with high-level FQ resistance or distribution in hot spots in the quinolone resistance-determining region (QRDR) of gyrA. Our findings provide essential implications for the feasibility of genotypic tests relying on detection of mutations in the QRDR of gyrA and the shorter first-line treatment regimens based on FQs in China.

Section: Discussioncontrasting

confidence: 55%

Prevalence and Molecular Characterization of Fluoroquinolone-Resistant Mycobacterium tuberculosis Isolates in China

Zhang

Wang

et al. 2014

“…In some instances, patients with TB may harbor more than one distinct bacterial population with possibly different susceptibility profiles (11,27). This appears to be particularly true in the case of FQ resistance (28)(29)(30)(31). It is possible that the remaining five FQ-resistant isolates with reportedly WT gyrA and gyrB QRDRs actually consist of wild-type susceptible M. tuberculosis mixed with a proportion of mutant cells that is below the level of detection by conventional sequencing.…”

Section: Resultsmentioning

confidence: 97%

Correlation between GyrA Substitutions and Ofloxacin, Levofloxacin, and Moxifloxacin Cross-Resistance in Mycobacterium tuberculosis

Willby

Sikes

Malik

et al. 2015

The newer fluoroquinolones moxifloxacin (MXF) and levofloxacin (LVX) are becoming more common components of tuberculosis (TB) treatment regimens. However, the critical concentrations for testing susceptibility of Mycobacterium tuberculosis to MXF and LVX are not yet well established. Additionally, the degree of cross-resistance between ofloxacin (OFX) and these newer fluoroquinolones has not been thoroughly investigated. In this study, the MICs for MXF and LVX and susceptibility to the critical concentration of OFX were determined using the agar proportion method for 133 isolates of M. tuberculosis. Most isolates resistant to OFX had LVX MICs of >1 g/ml and MXF MICs of >0.5 g/ml. The presence of mutations within the gyrA quinolone resistance-determining regions (QRDR) correlated well with increased MICs, and the level of LVX and MXF resistance was dependent on the specific gyrA mutation present. Substitutions Ala90Val, Asp94Ala, and Asp94Tyr resulted in low-level MXF resistance (MICs were >0.5 but <2 g/ml), while other mutations led to MXF MICs of >2 g/ml. Based on these results, a critical concentration of 1 g/ml is suggested for LVX and 0.5 g/ml for MXF drug susceptibility testing by agar proportion with reflex testing for MXF at 2 g/ml.I t is estimated that one-third of the world's population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), and 5% to 10% of infected persons will become ill with active disease necessitating a complicated treatment regimen. Patients with fully susceptible TB are generally treated with a combination of first-line drugs, including rifampin, isoniazid, ethambutol, and pyrazinamide for 6 months (1). Intolerance or acquisition of resistance to one or more of the first-line drugs requires deviation from the standard treatment regimen, and second-line drugs, including the injectables amikacin (AMK), kanamycin (KAN), and capreomycin (CAP) and one of a number of fluoroquinolone (FQ) derivatives, may be needed to successfully treat the patient.FQs are a class of broad-spectrum, synthetic antibiotics effective against many different bacterial infections such as those of the urinary and respiratory tracts, including infections by M. tuberculosis. Early FQs had a limited spectrum of activity; however, two of the newest FQ derivatives, moxifloxacin (MXF) and levofloxacin (LVX) (the levo isomer of ofloxacin [OFX]), exhibit superior bactericidal activity against a wide range of organisms, have longer half-lives, and are less toxic than older FQs (2, 3). Studies have demonstrated the effectiveness of these new FQs against M. tuberculosis (4, 5), with comparatively low MICs (MXF MIC Ͻ 0.5 g/ml) (1, 6-9). Although clinical resistance to FQs has been reported, estimating the global burden of FQ-resistant TB is difficult given that drug susceptibility testing (DST) is not performed in many areas and testing for FQ resistance is typically undertaken only when resistance to first-line drugs is detected. However, in 2013, the World Health Organization (WHO) estima...

“…Amino acid substitutions at other positions have not been reported, in strong contrast to the substitutions reported in M. tuberculosis, with predominant mutations in codon 94 (1,7,9,10,32,34,39), equivalent to codon 95 in M. leprae (Fig. 1).…”

Section: Discussionmentioning

confidence: 57%

“…To reveal the significance of amino acid substitution at position 95 to FQ resistance, we conducted the FQ-mediated supercoiling activity inhibition assay and DNA cleavage assay using recombinant DNA gyrases having an amino acid substitution in GyrA at position 95, Asp to Gly (GyrAAsp95Gly) or Asp to Asn (GyrA-Asp95Asn). These mutations are frequently found in FQ-resistant M. tuberculosis strains (1,7,9,10,32,34,39) but not in FQ-resistant M. leprae strains. …”

mentioning

confidence: 99%

Amino Acid Substitutions at Position 95 in GyrA Can Add Fluoroquinolone Resistance to Mycobacterium leprae

Yokoyama

Kim

Mukai

et al. 2012

Recently, fluoroquinolones (FQs), especially ofloxacin (OFX), have been recommended for the treatment of leprosy with a single lesion. The emergence of multidrug-resistant (MDR) leprosy, resistant to both DDS and RIF owing to therapeutic failure or low compliance, has been reported (17, 29), and FQs are thought to be important. For appropriate treatment, early assessment of drug susceptibility is essential; however, M. leprae cannot be cultivated on artificial media and a drug susceptibility test depending on in vitro growth is not available. Consequently, antibiotic susceptibility tests have relied on the mouse footpad leprosy model, requiring 8 to 12 months because of the slow growth of M. leprae (18). Recently, genetic analysis of drug-resistant M. leprae substantiated the correlation of DDS, RIF, and OFX resistance with mutations in folP1, encoding dihydropteroate synthetase (5, 15, 19, 23-25, 35); rpoB (4, 6, 12, 19, 23-25, 33), encoding the beta subunit of RNA polymerase; and gyrA, encoding the A subunit of DNA gyrase (4,19,24,26,40), respectively. Among these, data for folP1 in M. tuberculosis are not available as DDS is not used for the treatment of tuberculosis. Mutations in rpoB observed in M. leprae showed good agreement with those obtained from RIF-resistant M. tuberculosis. In contrast, the distribution of mutations in gyrA of FQresistant M. tuberculosis was distinct from that in gyrA of OFXresistant M. leprae (Fig. 1). Namely, amino acid substitutions at position 94 in GyrA were found in approximately half of FQ-resistant M. tuberculosis isolates, whereas no amino acid substitutions at position 95, equivalent to position 94 in M. tuberculosis, have been reported in M. leprae, and 11 cases with amino acid substitutions at position 91, equivalent to position 94 in M. tuberculosis, were reported from a total of six countries (4,19,24,26,40). Thus, elucidation of the contribution of amino acid substitutions at position 95 of GyrA in M. leprae to FQ resistance is important for the gene-based detection of fluoroquinolone resistance.FQs inhibit type II DNA topoisomerases, DNA gyrase, and topoisomerase IV, which play crucial roles in DNA replication during cell division (8). As M. leprae has only DNA gyrase, this is the sole target of FQs. DNA gyrase, consisting of two GyrA and two GyrB subunits, catalyzes the negative supercoiling of the circular bacterial chromosome by cleaving double strands and passing the enwrapped DNA, followed by resealing the double strands (8, 13). To reveal the significance of amino acid substitution at position 95 to FQ resistance, we conducted the FQ-mediated supercoiling activity inhibition assay and DNA cleavage assay using recombinant DNA gyrases having an amino acid substitution in GyrA at position 95, Asp to Gly (GyrAAsp95Gly) or Asp to Asn (GyrA-Asp95Asn). These mutations are frequently found in FQ-resistant M. tuberculosis strains (1,7,9,10,32,34,39) but not in FQ-resistant M. leprae strains. MATERIALS AND METHODS Materials.The Thai-53 strain of M. leprae (22), maintain...